8-11-14-eicosatrienoic-acid and Pseudomonas-Infections

A feature shared by many inflammatory lung diseases is excessive neutrophilic infiltration. Neutrophil homing to airspaces involve multiple factors produced by several distinct cell types. Hepoxilin A(3) is a neutrophil chemoattractant produced by pathogen-infected epithelial cells that is hypothesized to facilitate neutrophil breach of mucosal barriers. Using a Transwell model of lung epithelial barriers infected with Pseudomonas aeruginosa, we explored the role of hepoxilin A(3) in neutrophil transepithelial migration. Pharmacological inhibitors of the enzymatic pathways necessary to generate hepoxilin A(3), including phospholipase A(2) and 12-lipoxygenase, potently interfere with P. aeruginosa-induced neutrophil transepithelial migration. Both transformed and primary human lung epithelial cells infected with P. aeruginosa generate hepoxilin A(3) precursor arachidonic acid. All four known lipoxygenase enzymes capable of synthesizing hepoxilin A(3) are expressed in lung epithelial cell lines, primary small airway epithelial cells, and human bronchial epithelial cells. Lung epithelial cells produce increased hepoxilin A(3) and lipid-derived neutrophil chemotactic activity in response to P. aeruginosa infection. Lipid-derived chemotactic activity is soluble epoxide hydrolase sensitive, consistent with hepoxilin A(3) serving a chemotactic role. Stable inhibitory structural analogs of hepoxilin A(3) are capable of impeding P. aeruginosa-induced neutrophil transepithelial migration. Finally, intranasal infection of mice with P. aeruginosa promotes enhanced cellular infiltrate into the airspace, as well as increased concentration of the 12-lipoxygenase metabolites hepoxilin A(3) and 12-hydroxyeicosa-5Z,8Z,10E,14Z-tetraenoic acid. Data generated from multiple models in this study provide further evidence that hepoxilin A(3) is produced in response to lung pathogenic bacteria and functions to drive neutrophils across epithelial barriers.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 12-Lipoxygenase; Blood-Air Barrier; Epithelial Cells; Female; Humans; Male; Mice; Neutrophils; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Transendothelial and Transepithelial Migration

Eicosanoids are a group of bioactive lipids that are shown to be important mediators of neutrophilic inflammation; selective targeting of their function confers therapeutic benefit in a number of diseases. Neutrophilic airway diseases, including cystic fibrosis, are characterized by excessive neutrophil infiltration into the airspace. Understanding the role of eicosanoids in this process may reveal novel therapeutic targets. The eicosanoid hepoxilin A3 is a pathogen-elicited epithelial-produced neutrophil chemoattractant that directs transepithelial migration in response to infection. Following hepoxilin A3-driven transepithelial migration, neutrophil chemotaxis is amplified through neutrophil production of a second eicosanoid, leukotriene B4 (LTB4). The rate-limiting step of eicosanoid generation is the liberation of arachidonic acid by phospholipase A2, and the cytosolic phospholipase A2 (cPLA2)α isoform has been specifically shown to direct LTB4 synthesis in certain contexts. Whether cPLA2α is directly responsible for neutrophil synthesis of LTB4 in the context of

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Antigens, Human Platelet; Cell Communication; Cell Line; Chemotaxis; Coculture Techniques; Cystic Fibrosis; Cytosol; Humans; Leukotriene B4; Mice; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Mucosa; Tomography, Optical Coherence; Transendothelial and Transepithelial Migration

Recurrent Pseudomonas aeruginosa infections coupled with robust, damaging neutrophilic inflammation characterize the chronic lung disease cystic fibrosis (CF). The proresolving lipid mediator, 15-epi lipoxin A

Topics: 8,11,14-Eicosatrienoic Acid; Bacterial Proteins; Bronchoalveolar Lavage Fluid; Cell Line; Crystallography, X-Ray; Cystic Fibrosis; Humans; Inflammation; Lipoxins; Lung Diseases; Neutrophil Activation; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Retrospective Studies; Virulence Factors

Airway neutrophil infiltration is a pathological hallmark observed in multiple lung diseases including pneumonia and cystic fibrosis. Bacterial pathogens such as Pseudomonas aeruginosa instigate neutrophil recruitment to the air space. Excessive accumulation of neutrophils in the lung often contributes to tissue destruction. Previous studies have unveiled hepoxilin A(3) as the key molecular signal driving neutrophils across epithelial barriers. The eicosanoid hepoxilin A(3) is a potent neutrophil chemoattractant produced by epithelial cells in response to infection with P. aeruginosa. The enzyme phospholipase A(2) liberates arachidonic acid from membrane phospholipids, the rate-limiting step in the synthesis of all eicosanoids, including hepoxilin A(3). Once generated, aracidonic acid is acted upon by multiple cyclooxygenases and lipoxygenases producing an array of functionally diverse eicosanoids. Although there are numerous phospholipase A(2) isoforms capable of generating arachidonic acid, the isoform most often associated with eicosanoid generation is cytoplasmic phospholipase A(2)α. In the current study, we observed that the cytoplasmic phospholipase A(2)α isoform is required for mediating P. aeruginosa-induced production of certain eicosanoids such as prostaglandin E(2). However, we found that neutrophil transepithelial migration induced by P. aeruginosa does not require cytoplasmic phospholipase A(2)α. Furthermore, P. aeruginosa-induced hepoxilin A(3) production persists despite cytoplasmic phospholipase A(2)α suppression and generation of the 12-lipoxygenase metabolite 12-HETE is actually enhanced in this context. These results suggest that alterative phospholipase A(2) isoforms are utilized to synthesize 12-lipoxygenase metabolites. The therapeutic implications of these findings are significant when considering anti-inflammatory therapies based on targeting eicosanoid synthesis pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Base Sequence; Cell Line; Cytoplasm; Dinoprostone; Eicosanoids; Epithelial Cells; Group IV Phospholipases A2; Humans; Lung; Neutrophil Infiltration; Phospholipases A2; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Small Interfering; Transendothelial and Transepithelial Migration

Inflammation resulting from bacterial infection of the respiratory mucosal surface during pneumonia and cystic fibrosis contributes to pathology. A major consequence of the inflammatory response is recruitment of polymorphonuclear cells (PMNs) to the infected site. To reach the airway, PMNs must travel through several cellular and extracellular barriers, via the actions of multiple cytokines, chemokines, and adhesion molecules. Using a model of polarized lung epithelial cells (A549 or Calu-3) grown on Transwell filters and human PMNs, we have shown that Pseudomonas aeruginosa induces PMN migration across lung epithelial barriers. The process is mediated by epithelial production of the eicosanoid hepoxilin A(3) (HXA(3)) in response to P. aeruginosa infection. HXA(3) is a PMN chemoattractant metabolized from arachidonic acid (AA). Given that release of AA is believed to be the rate-limiting step in generating eicosanoids, we investigated whether P. aeruginosa infection of lung epithelial cells resulted in an increase in free AA. P. aeruginosa infection of A549 or Calu-3 monolayers resulted in a significant increase in [(3)H]AA released from prelabeled lung epithelial cells. This was partially inhibited by PLA(2) inhibitors ONO-RS-082 and ACA as well as an inhibitor of diacylglycerol lipase. Both PLA(2) inhibitors dramatically reduced P. aeruginosa-induced PMN transmigration, whereas the diacylglycerol lipase inhibitor had no effect. In addition, we observed that P. aeruginosa infection caused an increase in the phosphorylation of cytosolic PLA(2) (cPLA(2)), suggesting a mechanism whereby P. aeruginosa activates cPLA(2) generating free AA that may be converted to HXA(3), which is required for mediating PMN transmigration.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Cell Line; Cell Polarity; Chemotactic Factors; Cytosol; Enzyme Inhibitors; Epithelial Cells; Group IV Phospholipases A2; Humans; Lung; Male; Middle Aged; Neutrophil Infiltration; Phospholipases A; Phospholipases A2; Phosphorylation; Pseudomonas Infections